PRODUCTION OF ORYZACYSTATIN-I AND ORYZACYSTATIN-II IN ESCHERICHIA-COLI USING THE GLUTATHIONE-S-TRANSFERASE GENE FUSION SYSTEM

In recent years, several studies have demonstrated the potential of proteinase inhibitors for the biocontrol of insect pests. For such a control approach, however, strategies must be developed for the simple and efficient production of active inhibitors. In this study, oryzacystatins I (OCI) and II (OCII) were produced in Escherichia coli using the glutathione S-transferase (GST) gene fusion system. Both inhibitors were produced in large amounts as fusion products (similar to 100 mg/L E. coli culture) and were apparently stable when accumulated in bacterial cells. Enzyme specificities and inhibition constants of the fusion proteins were similar to those determined for free inhibitors and those reported for naturally occurring inhibitors isolated from rice, preventing the necessity of using an expensive and time-consuming cleavage step for the obtention of active OCs. The GST system thus appears appropriate for simple and efficient large-scale production of the two cysteine proteinase inhibitors.